U.S. patent number 8,173,377 [Application Number 13/016,409] was granted by the patent office on 2012-05-08 for methods and compositions for determining the purity of chemically synthesized nucleic acids.
This patent grant is currently assigned to North Carolina State University. Invention is credited to Paul F. Agris, Lloyd G. Mitchell, Christopher D. J. Pearce.
United States Patent |
8,173,377 |
Agris , et al. |
May 8, 2012 |
**Please see images for:
( Certificate of Correction ) ** |
Methods and compositions for determining the purity of chemically
synthesized nucleic acids
Abstract
This application describes an antibody that specifically binds
to a synthetic oligomer (e.g., an oligonucleotide or oligopeptide)
having a organic protecting group covalently bound thereto, which
antibody does not bind to that synthetic oligomer when the organic
protecting group is not covalently bound thereto. Methods of making
and using such antibodies are also disclosed, along with cells for
making such antibodies and articles carrying immobilized oligomers
that can be used in assay procedures with such antibodies.
Inventors: |
Agris; Paul F. (Raleigh,
NC), Pearce; Christopher D. J. (Surrey, GB),
Mitchell; Lloyd G. (Durham, NC) |
Assignee: |
North Carolina State University
(Raleigh, NC)
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Family
ID: |
23893992 |
Appl.
No.: |
13/016,409 |
Filed: |
January 28, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20110190154 A1 |
Aug 4, 2011 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10190795 |
Jul 8, 2002 |
7901892 |
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09747467 |
Dec 22, 2000 |
6929907 |
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09476975 |
Dec 31, 1999 |
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Current U.S.
Class: |
435/7.1;
435/7.2 |
Current CPC
Class: |
C07K
16/18 (20130101); C12Q 1/6804 (20130101); C07K
16/44 (20130101); Y02P 20/55 (20151101); B01J
2219/00274 (20130101) |
Current International
Class: |
G01N
33/53 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 906 917 |
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Apr 1999 |
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EP |
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11-80185 |
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Mar 1999 |
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JP |
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WO 98/03532 |
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Jan 1998 |
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WO |
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Other References
Andrews et al. "Analysis of DNA adducts using high-performance
separation techniques coupled to electrospray ionization mass
spectrometry" Journal of Chromatography, vol. 856, No. 1-2, (Sep.
24, 1999), pp. 515-526. cited by other .
Chersi et al. "Preparation of Rabbit Antibodies to 4 4'
Dimethoxytriphenylmethyl the Prosepective Goup in Oligonucleotide
Synthesis" Biological Chemistry Hoppe-Seyler, v. 372, No. 9, 1991,
pp. 845-848. cited by other .
Degling, Lena, et al., Biodegradable microspheres XVIII: the
adjuvant effect of polyacryl starch microparticles with conjugated
human serum albumin, Vaccine, vol. 13, No. 7, pp. 629-636, 1995.
cited by other .
European Search Report for Application No. PC/JM/P12326EP; Dated
Nov. 5, 2004. cited by other .
Fodor S P A et al. Light-directed, spatially addressable parallel
chemical synthesis. Science. Feb. 15, 1991;251(4995):767-773. cited
by other .
Hashizume et al: "Specificity of anti-polynucleotide monoclonal
antibodies from human-human hybridomas." In Vitro Cellular &
Developmental Biology: Journal of the Tissue Culture Association,
v. 23, No. 1, 1987, pp. 53-56. cited by other .
International Search Report, International Application No.
PCT/US00/35600 dated Apr. 17, 2001. cited by other .
Partial European Search Report, EP 08 00 6268, Jun. 5, 2008. cited
by other .
Schena et al. PNAS 1996 vol. 93, p. 10614-10619. cited by other
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Supplemental Partial European Search Report, EP 00 99 0402 mailed
May 11, 2004. cited by other .
Tortora et al. Microbiology 6.sup.th Edition (1997) Addison Wesley
Longman, Inc. p. 497. cited by other .
Weiler et al. Combining the preparation of oligonucleotide arrays
and synthesis of high-quality primers. Analytical Biochemistry.
Dec. 15, 1996;243(2):218-227. cited by other.
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Primary Examiner: Cheu; Jacob
Attorney, Agent or Firm: Myers Bigel Sibley & Sajovec,
P.A.
Parent Case Text
RELATED APPLICATIONS
This application is a divisional of and claims priority to U.S.
patent application Ser. No. 10/190,795, filed Jul. 8, 2002, now
U.S. Pat. No. 7,901,892, which is a continuation of U.S. patent
application Ser. No. 09/747,467, filed Dec. 22, 2000, now U.S. Pat.
No. 6,929,907, which is a continuation-in-part of U.S. patent
application Ser. No. 09/476,975, filed Dec. 31, 1999, abandoned,
the disclosure of each of which is incorporated by reference herein
in its entirety.
Claims
That which is claimed is:
1. A method of using an oligonucleotide array and compensating for
insufficient deprotection or insufficient elongation of
oligonucleotides on said array, comprising the steps of: (a)
providing a substrate having a plurality of different
oligonucleotides immobilized thereon, with said different
oligonucleotides immobilized in different separate and discrete
locations on said substrate; (b) providing indicia associated with
said array, said indicia recording the presence of insufficient
deprotection or insufficient elongation of at least one
oligonucleotide, said at least one oligonucleotide located in a
separate and discrete locations on said array; (c) providing a test
compound; (d) detecting the binding of said test compound to at
least one of said plurality of different oligonucleotides; and then
(d) determining the degree of binding of said test compound to said
oligonucleotide from (i) said detected binding and (ii) said
indicia recording the presence of insufficient deprotection or
insufficient elongation, so that said insufficient deprotection or
insufficient elongation is compensated for during said determining
step.
2. A method according to claim 1, wherein said test compound is a
protein, peptide, or oligonucleotide.
3. A method according to claim 1, wherein said test compound is
mRNA.
4. A method according to claim 1, wherein said determining step is
carried out by generating a color indication of degree of
binding.
5. A method according to claim 1, wherein said determining step is
carried out by generating a numeric indication of degree of
binding.
6. A method according to claim 1, wherein said degree of binding is
binding affinity, binding amount, or both binding affinity and
binding amount.
7. A method of using an oligonucleotide array while compensating
for insufficient deprotection or insufficient elongation of
oligonucleotides on said array, said method comprising the steps
of: (a) providing a substrate having a plurality of different
oligonucleotides immobilized thereon, with said different
oligonucleotides immobilized in different separate and discrete
locations on said substrate; (b) providing indicia associated with
said array, said indicia recording the presence of insufficient
deprotection or insufficient elongation of at least one
oligonucleotide, said at least one oligonucleotide located in a
separate and discrete locations on said array; (c) providing a test
compound; (d) contacting said test compound to said array; (d)
deleting from analysis said at least one oligonucleotide in a
separate and discrete location having insufficient deprotection,
with binding of said test compound to said array being detected
with the remaining oligonucleotides in separate and discrete
locations that have not been deleted from analysis; and then (d)
detecting the binding of said test compound to said remaining
oligonucleotides in separate and discrete locations in said
array.
8. A method according to claim 7, wherein said test compound is a
protein, peptide, or oligonucleotide.
9. A method according to claim 7, wherein said test compound is
mRNA.
10. A method according to claim 7, wherein said detecting step is
carried out by generating a color indication of binding.
11. A method according to claim 7, wherein said detecting step is
carried out by generating a numeric indication of binding.
Description
FIELD OF THE INVENTION
The present invention concerns the detection, identification and
quantification of protecting groups remaining after chemical
synthesis of oligomers, particularly oligonucleotides.
BACKGROUND OF THE INVENTION
Over the past decade automated chemical synthesis of nucleic acids
such as DNA and RNA on solid supports has been developed. These
chemical processes include the use of agents to protect the
exocyclic amines of the nucleotide bases adenine, thymine, cytosine
and guanine and to direct the synthesis by blocking the 2'OH of
RNA's ribose. The bases within the nucleic acid product of the
synthesis are deprotected upon cleavage of the nucleic acid from
the solid support. However, the extent of base deprotection is not
easily determined.
For example, after base deprotection of synthetic RNA, products
still contain the 2'-dimethylsilyl tert-butyl group as a protection
of the 2'OH of the ribose moiety. This protecting group is removed
carefully by chemical means so as not to effect the chemistry and
structure of the RNA. However, the extent of deprotection of the
2'OH is not readily determined. The nucleic acid is purified by
high pressure liquid chromatography or by gel electrophoresis.
However, some of the unwanted products of the synthesis are
complete nucleic acid sequences that still contain one or more
protecting groups, and shorter than full length (aborted) sequences
difficult to separate from full length sequences, especially for
oligomers of longer than 50 nucleosides. At present, there is no
easy method to determine how much of each protecting group, if any,
still remains on the product, and what proportion of the product is
full-length. See generally Davis, G. E., Gehrke, C. W., Kuo, K. C.,
and Agris, P. F. (1979) Major and Modified Nucleosides in tRNA
Hydrolysates by High Performance Liquid Chromatography. J.
Chromatogr. 173:281-298; Agris, P. F., Tompson, J. G., Gehrke, C.
W., Kuo, K. C., and Rice, R. H. (1980) High-Performance Liquid
Chromatography and Mass Spectrometry of Transfer RNA Bases for
Isotopic Abundance. J. Chromatogr. 194:205-212; Gehrke, C. W., Kuo,
K. C., McCune, R. A., Gerhardt, K. O., and Agris, P. F. (1981)
Quantitative Enzymatic Hydrolysis of tRNAs: RP-HPLC of tRNA
Nucleosides. J. Chromatogr. 230:297-308; Chromatography and
Modification of Nucleosides Volumes A, B and C (Gehrke, C. W. and
Kuo, K. C. T., eds.), Elsevier Publishing Co. 1990; Agris, P. F.
and Sierzputowska-Gracz, H. (1990) Three Dimensional Dynamic
Structure of tRNA's by Nuclear Magnetic Resonance. In
Chromatography and Modification of Nucleosides (Gehrke, C. W. and
Kuo, K. C. T., eds.), Elsevier Publishing Co., pp. 225-253; Agris,
P. F., Hayden, J., Sierzputowska-Gracz, H., Ditson, S., Degres, J.
A., Tempesta, M., Kuo, K. C. and Gehrke, C. W. (1990) Compendium on
Biological, Biochemical, Chemical, Physical and Spectroscopic
Properties of RNA and DNA Nucleosides. In Chromatography and
Modification of Nucleosides, Elsevier Publishing Co.
The incomplete removal of the protecting group and lack of a simple
assay is a problem for two industries and for numerous researchers
world wide: (i) the multitude of companies now providing nucleic
acid sequence synthesis products by overnight delivery have
difficulty telling their customers the extent to which the product
is deprotected; (ii) pharmaceutical companies cannot easily verify
for regulatory agencies the purity and/or length of the therapeutic
or diagnostic oligonucleotide products they seek to introduce or
market. Accordingly, there is a need for simple and reliable
techniques for determining the purity and proportion of full length
of oligonucleotide products.
SUMMARY OF THE INVENTION
A first aspect of the present invention is an antibody (e.g., a
monoclonal or polyclonal antibody) that specifically binds to a
synthetic oligomer (i.e., an oligonucleotide or oligopeptide)
having a organic protecting group covalently bound thereto, which
antibody does not bind to that synthetic oligomer when the organic
protecting group is not covalently bound thereto.
A second aspect of the present invention comprises a cell or cells,
including cell cultures and isolated cells, that express an
antibody as described above. Such cells include hybridoma cells, as
well as recombinant cells that contain and express a heterologous
nucleic acid encoding the antibody.
A third aspect of the present invention is a method for detecting
incomplete deprotection of a synthetic oligomer by immunoassay,
said immunoassay comprising the steps of: (a) contacting a
synthetic oligomer to an antibody as described above, and then (b)
detecting the presence or absence of binding of said antibody to
said oligomer, the presence of binding indicating incomplete
deprotection of said synthetic oligomer. Any suitable assay format
can be employed, including heterogeneous and homogeneous
immunoassays. For example, the immunoassay may be an immunoblot-dot
assay, or may be a sandwich assay.
A fourth aspect of the present invention is a method for separating
protected (including partially and completely protected) synthetic
oligomers from fully deprotected synthetic oligomers. The method
comprises (a) contacting a mixture of protected from fully
deprotected synthetic oligomers to antibodies as described above,
wherein the protected synthetic oligomers have the organic
protecting group covalently bound thereto, so that the protected
synthetic oligomers bind to the antibody; and then separating the
antibodies from the fully deprotected oligomers. The antibody may
be immobilized on a solid support to facilitate separation. The
protected synthetic oligomer may be a partially protected synthetic
oligomer (for which one application is the identification and/or
purification of full-length versus aborted sequence oligomers) or a
fully protected synthetic oligomer that has not undergone
deprotection. Any separation format may be used, including but not
limited to affinity chromatography.
A fifth aspect of the invention is an article useful for the
determining incomplete deprotection of a synthetic oligomer in an
immunoassay, said article comprising: (a) a solid support (e.g., a
nitrocellulose strip) having a surface portion, said surface
portion having at least two separate discrete regions formed
thereon; (b) a first oligomer bound to one of said separate
discrete regions, said first oligomer having a protecting group
bound thereto; and (c) a second oligomer bound to another of said
separate discrete regions, said second oligomer not having said
protecting group bound thereto; wherein the nucleotide sequence of
said first and second oligomers are the same. In a preferred
embodiment, the article further comprises (d) a third oligomer
bound to another of said separate discrete regions; said third
oligomer also having said protecting group bound to said first
oligomer bound thereto; wherein said third oligomer is partially
deprotected; and wherein the nucleotide sequence of said first,
second, and third oligomers are the same.
A sixth aspect of the present invention is a method of making an
antibody that specifically binds to a synthetic oligomer having a
organic protecting group covalently bound thereto, which antibody
does not bind to the said synthetic oligomer when said organic
protecting group is not covalently bound thereto, said method
comprising the steps of: (a) synthesizing said synthetic oligomer
on a solid particulate support (and preferably covalently bound
thereto, e.g., with a succinyl linker) with said organic protecting
group covalently bound to said synthetic oligomer (or synthesizing
a monomer of a single nucleotide on the solid support, with the
single nucleotide having said protecting group covalently bound
thereto); and then, without removing said oligomer from said solid
support; (b) immunizing an animal with said synthetic oligomer
bound to said solid support (or monomer bound to said solid
support) in an amount sufficient to produce said antibody.
Optionally, the solid support can be replaced with a carrier group
such as a protein (e.g., bovine serum albumin).
In summary, the antibodies and methods of the present invention are
useful in immunoassays, such as for the qualitative and
quantitative detection of protecting groups used in organic
synthetic processes, with particular application to
oligonucleotides or peptides in research, therapeutics, diagnostics
and biomedical science. The antibodies of the invention can be used
in purification techniques, such as for the separation of final
products from by-product contaminants. The instant invention can be
used in the course of quality control of oligonucleotide and
peptide synthesis, such as in the quality control of drugs for gene
therapy, antisense, antigene and control of gene expression, in the
quality control of biomedical polymers that may contain protecting
groups, and as probes for purification and characterization of
synthetic oligomers, particularly oligonucleotides or peptides.
The present invention is explained in greater detail in the
drawings herein and the specification set forth below.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a dot-blot immunoassay of monoclonal antibody 1H11, which
selectively binds to oligoIbu-dG20mers.
FIG. 2 is a dot-blot immunoassay of monoclonal antibody 7H3, which
selectively binds to oligoBz-dC20mers.
FIG. 3 shows ELISA (A) and dot-blot (B) results demonstrating
specificity and detection sensitivity of a monoclonal antibody
(mAb) of the commonly used protecting group, benzoyl (Bz), for the
chemical synthesis of nucleic acids. Partially deprotected oligomer
oligo Bz-dC (center column) can be re-treated to remove the
remaining protecting groups, and re-tested with mAb (C). An RNA
standard with protecting groups Bz, ibu and ipr-Pac was synthesized
and assayed for identification of the protecting groups with the
mAb against Bz (D).
FIG. 4 shows ELISA (A) and dot-blot (B) results demonstrating
specificity and sensitivity of a monoclonal antibody (mAb) and its
detection of the commonly used protecting group, isobutryl (ibu),
for the chemical synthesis of nucleic acids. Dot-blot assay with
high amounts of DNA demonstrates that the ibu protecting group was
recognized by the mAb no matter which nucleobase was protected (C).
Partially deprotected oligomer oligo Bz-dC (center column) can be
re-treated to remove the remaining protecting groups, and re-tested
with mAb (D). An RNA standard with protecting groups Bz, ibu and
ipr-Pac was synthesized and assayed for identification of the
protecting groups with the mAb against ibu (E).
FIG. 5 shows ELISA (A) and dot-blot (B) results demonstrating
specificity and sensitivity of a monoclonal antibody (mAb) and its
detection of the commonly used protecting group,
isopropylphenoxyacetyl (ipr-Pac), for the chemical synthesis of
nucleic acids. Partially deprotected oligomers oligo ibr-Pac-dG and
oligo ibu-dG (columns second from left and forth from left,
respectively) can be re-treated to remove the remaining protecting
groups, and re-tested with mAb (C). An RNA standard with protecting
groups Bz, ibu and ipr-Pac was synthesized and assayed for
identification of the protecting groups with the mAb against
ipr-Pac (D).
FIG. 6 shows a mAb dot-blot assay of protecting groups
demonstrating the sensitivity and quantifiable response of the
technology as related to HPLC. Dot-blot detection of Bz groups
remaining on a standardized 20mer oligo dC molecule was analyzed
(A) and a quantitation of the mAb response (B) was determined. The
mAb response was analyzed with an increase in the amount of DNA on
the dot-blot membrane (C). The column on the left is just the
protected Bz-dC 20mer. The column on the right is the protected
Bz-dC together with a 2500-fold excess of the completely
deprotected oligo dC(Bz).
FIG. 7 shows a direct comparison of the mAb and HPLC detection of
Bz in the pmole (A) and nmol range (B), respectively.
FIG. 8 shows a blind study demonstrating the detection of remaining
protecting groups in commercial samples. dA-dC oligos were analyzed
with anti-Bz mAb (A) and dG-dT oligos were analyzed with
anti-ipr-Pac mAb (B). The oligo dA-dC samples from companies #2 and
#6 were tested in higher amounts to confirm the presence of the Bz
protecting group (C). In addition, the samples were treated to
remove the remaining protecting groups using a standard protocol.
The oligo dG-dT samples were assayed for the ipr-Pac protecting
groups (D). The samples were re-treated to remove remaining
protecting groups and re-analyzed as in (C).
FIG. 9 shows the production and analyses of polycolonal antibody
against the 5' terminal protecting group, dimethyltrityl (DMT).
FIG. 10 shows a substrate carrying different oligonucleotides of
the same sequence, but with varying degrees of deprotection, that
may be used as a testing standard to screen similar
oligonucleotides of the same sequence for varying degrees of
protection or deprotection.
FIG. 11 illustrates an oligonucleotide array that may be screened
for the presence of protecting groups or insufficient elongation
with antibodies of the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. General Definitions.
"Antibody" as used herein refers to both monoclonal and polyclonal
antibodies, refers to antibodies of any immunoglobulin type
(including but not limited to IgG and IgM antibodies), and
including antibody fragments that retain the hypervariable or
binding regions thereof. Antibodies may be of any species of
origin, but are typically mammalian (e.g., horse, rat, mouse,
rabbit, goat). Antibodies may be bound to or immobilized on solid
supports such as nitrocellulose, agarose, glass, organic polymers
("plastics") and the like in accordance with known techniques, and
may be labeled with or joined to other detectable groups in
accordance with known techniques.
"Binding" as used herein with respect to the selective binding of
an antibody to an oligomer has its usual meaning in the art. In
general, to obtain useful discrimination in an immunoassay or an
affinity purification technique, the antibody should bind to the
protected oligomer at an affinity of at least about
k.sub.d=10.sup.-6, 10.sup.-7, or 10.sup.-8 M, and should bind to
the unprotected oligomer at an affinity of not greater than about
k.sub.d=10.sup.-2, 10.sup.-3, or 10.sup.-4 M.
"Oligomer" as used herein refers to synthetic oligonucleotides and
synthetic oligopeptides, including synthetic oligomers in the
naturally occurring form such as DNA and RNA, and modified backbone
chemistries as discussed below. Oligonucleotides are currently
preferred in carrying out the present invention, and the instant
invention is primarily explained with reference to oligonucleotides
herein. However, the methods and techniques described herein may
also be applied to oligopeptides, oligosaccharides, etc. (i.e., any
synthetically produced polymer requiring protecting groups for
synthesis).
"Nucleotide" as used herein refers to a subunit of an
oligonucleotide comprising a pentose, a nitrogenous heterocyclic
base (typically bound to the 1 position of the pentose), and a
phosphate or phosphoric acid group (typically bound at the 5'
position of the pentose) but absent, or considered bound at the 3'
position, in the 5' terminal nucleotide of an oligonucleotide.
These structures are well known. See, e.g., A. Lehninger,
Biochemistry, 309-320). "Nucleoside" typically refers to a
nucleotide, absent a phosphoric acid or phosphate group.
"Protecting group" as used herein has its conventional meaning in
the art and refers to a chemical moiety, group or substituent that
is coupled, typically covalently coupled, to an atom in a molecule
prior to a chemical reaction involving that molecule (typically in
an organic synthesis), so that the chemical reaction is averted at
the atom to which the protecting group is coupled. Typically, the
protecting group is then chemically removed from the intermediate
molecule for preparation of the final product, although removal
techniques may not be entirely successful leading to only partial
deprotection of the final product (i.e., the presence of at least
one protecting group remaining on that molecule). Protecting groups
may be intentionally left on a molecule for purposes of generating
or testing an antibody as described herein.
"Deprotection" or "deprotected" as used herein refers to the
absence of protecting groups employed during chemical
oligonucleotide synthesis from a molecule. Such protecting groups
are described below. The presence of such a protecting group may
indicate insufficient elongation of the oligonucleotide, when the
protecting group is chain terminating. Chemically synthesized
oligonucleotides are ideally fully deprotected, but the present
invention is employed to detect partial or incomplete deprotection
of such oligonucleotides (that is, the presence of at least one
protecting group as described below in the oligonucleotide).
"Base" as used herein with respect to oligonucleotides refers to a
nitrogenous heterocyclic base which is a derivative of either
purine (e.g., adenine, guanine) or pyrimidine (e.g., uracil,
thymine, cytosine). Pyrimidine bases are bound to the pentose by
the 1 ring nitrogen; Purine bases are bonded to the pentose by the
9 ring nitrogen. Preferred bases are those that contain a free
amino group, such as guanine, adenine, and cytosine (the protecting
group is then covalently bound to the free amino group by
substitution of one, or both, of the hydrogens on the free amino
group). However, the present invention may be used with any purine
or pyrimidine base, whether standard or modified/rare, that
contains a free amino group for protection, or other group
requiring protection during synthesis thereof in an
oligonucleotide. Examples of standard and modified/rare bases are
those found in the nucleosides set forth in Table 1 below.
TABLE-US-00001 TABLE 1 Standard and modified nucleosides and their
standard abbreviations. abbreviation base U uridine C cytidine A
adenosine G guanosine T thymidine ?A unknown modified adenosine m1A
1-methyladenosine m2A 2-methyladenosine i6A
N.sup.6-isopentenyladenosine ms2i6A
2-methylthio-N.sup.6-isopentenyladenosine m6A
N.sup.6-methyladenosine t6A N.sup.6-threonylcarbamoyladenosine
m6t6A N.sup.6-methyl-N.sup.6-threonylcarbomoyladenosine ms2t6A
2-methylthio-N.sup.6-threonylcarbamoyladenosine Am
2'-O-methyladenosine I Inosine m1I 1-methylinosine Ar(p)
2'-O-(5-phospho)ribosyladenosine io6A
N.sup.6-(cis-hydroxyisopentenyl)adenosine ?C Unknown modified
cytidine s2C 2-thiocytidine Cm 2'-O-methylcytidine ac4C
N.sup.4-acetylcytidine m5C 5-methylcytidine m3C 3-methylcytidine
k2C lysidine f5C 5-formylcytidine f5Cm 2'-O-methyl-5-formylcytidine
?G unknown modified guanosine Gr(p)
2'-O-(5-phospho)ribosylguanosine m1G 1-methylguanosine m2G
N.sup.2-methylguanosine Gm 2'-O-methylguanosine m22G
N.sup.2N.sup.2-dimethylguanosine m22Gm
N.sup.2,N.sup.2,2'-O-trimethylguanosine m7G 7-methylguanosine
fa7d7G archaeosine Q queuosine manQ mannosyl-queuosine galQ
galactosyl-queuosine Yw wybutosine o2yW peroxywybutosine ?U unknown
modified uridine mnm5U 5-methylaminomethyluridine s2U 2-thiouridine
Um 2'-O-methyluridine s4U 4-thiouridine ncm5U
5-carbamoylmethyluridine mcm5U 5-methoxycarbonylmethyluridine
mnm5s2U 5-methylaminomethyl-2-thiouridine mcm5s2U
5-methoxycarbonylmethyl-2-thiouridine cmo5U uridine 5-oxyacetic
acid mo5U 5-methoxyuridine cmnm5U 5-carboxymethylaminomethyluridine
cmnm5s2U 5-carboxymethylaminomethyl-2-thiouridine acp3U
3-(3-amino-3-carboxypropyl)uridine mchm5U
5-(carboxyhydroxymethyl)uridinemethyl ester cmnm5Um
5-carboxymethylaminomethyl-2'-O-methyluridine ncm5Um
5-carbamoylmethyl-2'-O-methyluridine D Dihydrouridine .psi.
pseudouridine m1.psi. 1-methylpseudouridine .psi.m
2'-O-methylpseudouridine m5U ribosylthymine m5s2U
5-methyl-2-thiouridine m5Um 5,2'-O-dimethyluridine
See Sprinzl et al., Nucleic Acids Res. 26, 148 (1998).
Applicants specifically intend that the disclosures of all United
States patent references cited herein be incorporated by reference
herein in their entirety.
2. Protecting Groups.
The particular protecting group will depend upon the oligomer being
synthesized and the methodology by which that oligomer is
synthesized.
For the synthesis of oligonucleotides, suitable protecting groups
include alkyl, aryl, alkylaryl, arylalkyl groups, which may contain
one or more hetero atoms such as N, O, or S, and which may be
substituted or unsubstituted (e.g., a carbonyl group). Examples of
protecting groups include, but are not limited to, the following:
acetyl; isobutyryl; 2-(t-butyldiphenyl-silyloxymethyl)benzoyl;
naphthaloyl; isobutyryloxycarbonyl; levulinyl;
fluorenylmethoxycarbonyl; 2-nitrothiophenyl;
2,2,2-trichloro-t-butoxycarbonyl, ethoxycarbonyl;
benzyloxycarbonyl; p-nitrophenylethyloxycarbonyl;
N'N-dimethylformamidine; formyl; benzoyl, toluoyl;
2,4-6-trimethylbenzoyl; anisoyl; 2,4-dimethylphenyl;
2,4,6-trimethylphenyl; triphenylthiomethyl; pivoloiloxymethyl;
t-butoxycarbonyl; p-nitrophenylethyl; methoxyethoxymethyl;
butylthiocarbonyl; 2-methyl-pyridine-5-yl; 2-nitrothiophenyl;
2,4-dinitrothiophenyl; 2-nitro-4-methylthiophenyl;
p-nitrophenylsulphonylethyl; 5-chloro-8-hydroxyquinoline;
thiophenyl; .beta.-cyanoethyl; phenylethyl; p-nitrophenylethyl;
pyridylethyl; 2-N-methylimidazolylphenyl; methyl; allyl;
trichloroethyl; dibenzoyl; p-nitrophenylethoxycarbonyl; benzoyl and
substituted derivatives thereof; 2(acetoxymethyl) benzoyl;
4,4',4''-tris-(benzyloxy)trityl; 5-methylpyridyno-2-yl;
phenylthioethyl; dipehylcarbamoyl; 3,4-dimethoxybenzyl;
3-chlorophenyl; 2-nitrophenyl; 9-pnenylxanthen-9-yl;
9-(p-methoxyphenyl)xanthen-9-yl;
9-(p-ocatadecyloxyphenyl)xanthen-9-yl; "bridged"
bis-dimethoxytrityl groups; phthaloyl; succinyl;
benzensulphonylethoxycarbonyl; 4,4',4''-tris(bevulinyloxy)trityl;
p-phenylazophenyloxycarbonyl; o-substituted benzoyl;
4,4'4''-tris-(4,5-dichlorophalimidin)trityl; levelinyl; alkyloxy
and aryloxyacetyl; 1,3-benzodithiol-2-yl; tetrahydrofuranyl;
[2-(methylthio)phenyl]thiomethyl; 1-(2-chloroethyoxy)ethyl;
1-[(2-fluoro-phenyl]4-methoxy piperidin-4-yl;
4-methoxytetrahydropyran-4-yl; (1-methyl-1-methoxy)ethyl;
tetrahydropyranyl; 3-methoxy-1,5-dicarbomethoxypentam-3-yl;
2-nitrobenzyl; benzyl; 4-nitrophenylethyl-sulphonyl;
t-butyldimethylsilyl; 4-methoxybenzyl; 3,4-dimethoxybenzyl;
9-p-methoxyphenylthioxanthen-9-yl; compounds of the formula
R.sub.1R.sub.2R.sub.3C--, wherein R.sub.1, R.sub.2, and R.sub.3 are
each independently selected from the group consisting of phenyl,
p-monomethoxyphenyl, o-monomethoxphenyl, biphenyl, p-fluoropnehyl,
p-chlorophenyl, p-methylphenyl, p-nitrophenyl, etc.
3. Oligonucleotides.
Synthetic oligonucleotides that contain protecting groups and may
be used to carry out the present invention include both the
naturally occurring forms such as DNA and RNA, and those with
modified backbone chemistries, such as poly (phosphate derivatives)
such as phosphonates, phosphoramides, phosphonamides, phosphites,
phosphinamides, etc., poly (sulfur derivatives) e.g., sulfones,
sulfonates, sulfites, sulfonamides, sulfenamides, etc. It will be
noted that antibodies of the invention may be characterized by
their selective binding to particular "reagent" or "benchmark"
oligonucleotides, but the same antibodies may also bind to a
variety of other oligonucleotides (e.g., longer nucleotides) or
other compounds that contain the same protecting group.
For example, an oligonucleotide to which the antibody selectively
binds may consist of from 3 to 20 nucleotides, and wherein one of
said nucleotides is a protected nucleotide according to Formula (I)
below:
##STR00001## wherein:
R is H or a protecting group, such as dimethoxytrityl; subject to
the proviso that R is a covalent bond to an adjacent nucleotide
when said protected base is not a 5' terminal nucleotide in said
oligonucleotide;
R.sub.1 is H or a protecting group such as .beta.-cyanoethyl;
subject to the proviso that R.sub.1 is a covalent bond to an
adjacent nucleotide when said protected base is not a 3' terminal
nucleotide in said oligonucleotide;
R.sub.2 is H or --OR.sub.3;
R.sub.3 is H or a protecting group such as
tert-butyldimethylsilyl;
Base is a purine or pyrimidine base; and
R.sub.4 is a protecting group bonded to an amino group of said
base, such as a protecting group is selected from the group
consisting of acetyl (Ac), benzoyl (Bz), dimethylformamidine (dmf),
isobutyrl (Ibu), phenoxyacetyl (Pac), and isopropyl-phenoxyacetyl
(Ipr-pac);
and further subject to the proviso that when one of R, R.sub.1,
R.sub.3 and R.sub.4 is a protecting group, then the others of R,
R.sub.1, R.sub.3 and R.sub.4 are not protecting groups.
In one particular embodiment of the foregoing, the antibody may be
one that selectively binds to an oligonucleotide that consists of
from 3 to 20 nucleotides and has a 5' nucleotide, and wherein said
5' nucleotide is a protected nucleotide according to Formula
(I):
##STR00002## wherein:
R is a protecting group such as dimethoxytrityl;
R.sub.1 is a covalent bond to an adjacent nucleotide;
R.sub.2 is --H or --OH; and
Base is a purine or pyrimidine base.
In another particular embodiment of the foregoing, the antibody may
be one that selectively binds to an oligonucleotide that consists
of from 3 to 20 nucleotides and has a 3' nucleotide, and wherein
said 3' nucleotide is a protected nucleotide according to Formula
(I):
##STR00003## wherein:
R is a covalent bond to an adjacent nucleotide;
R.sub.1 is a protecting group such as .beta.-cyanoethyl;
R.sub.2 is H or --OH; and
Base is a purine or pyrimidine base.
In another particular embodiment of the foregoing, the antibody may
be one that selectively binds to an oligonucleotide that consists
of from 3 to 20 nucleotides, and wherein one of said nucleotides is
a protected nucleotide according to Formula (I):
##STR00004## wherein:
R is a covalent bond to an adjacent nucleotide;
R.sub.1 is a covalent bond to an adjacent nucleotide;
R.sub.2 is --OR.sub.3;
R.sub.3 a protecting group such as tert-butyldimethylsilyl; and
Base is a purine or pyrimidine base.
In still another particular embodiment of the foregoing, the
antibody may be one that selectively binds to an oligonucleotide
that consists of from 3 to 20 nucleotides, and wherein one of said
nucleotides is a protected nucleotide according to Formula (I):
##STR00005## wherein:
R is a covalent bond to an adjacent nucleotide;
R.sub.1 is a covalent bond to an adjacent nucleotide;
R.sub.2H or --OH;
Base is a purine or pyrimidine base; and
R.sub.4 is a protecting group bonded to an amino group of said
base, such as acetyl, benzoyl, dimethylformamidine, isobutyryl,
phenoxyacetyl, and isopropyl-phenoxyacetyl.
Thus, examples of protected bases that may be employed in the
structures shown above include, but are not limited to, adenine,
guanine, and cytosine, as follows:
##STR00006## wherein R.sub.1 and R.sub.2 are both H in an
unprotected base, and either R.sub.1 or R.sub.2 are a protecting
group as described above (e.g. Pac, Ipr-pac, Ibu, Bz, Ac, dmf) for
a protected base. Likewise, modified nucleosides have protecting
groups at the modifications that are chemically reactive.
In one embodiment of the invention, the oligonucleotides are
peptide nucleic acids, and the protecting groups are those
protecting groups employed in the synthesis of peptide nucleic
acids, including but not limited to those described in U.S. Pat.
No. 6,133,444.
In still another particular embodiment of the foregoing, the
antibody may be one that selectively binds to an oligonucleotide
that consists of from 3 to 20 nucleotides, and wherein one of said
nucleotides is a protected with a photolabile protecting group,
including but not limited to those described in U.S. Pat. Nos.
5,744,101 and 5,489,678 (assigned to Affymax).
4. Antibodies.
As noted above, the present invention provides antibodies (e.g., a
monoclonal or polyclonal antibody) that specifically bind to a
synthetic oligonucleotide having a organic protecting group
covalently bound thereto, which antibody does not bind to said
synthetic oligonucleotide when said organic protecting group is not
covalently bound thereto.
The antibody may be provided immobilized on (or bound to) a solid
support in accordance with known techniques, or may be provided in
a free, unbound form (e.g., lyophilized, frozen, in an aqueous
carrier, etc.). Whether or not an antibody is immobilized will
depend upon the particular immunoassay or affinity purification
technique in which the antibody is used, and is determined by the
known parameters for such techniques. Similarly, the antibody may
be bound to or conjugated with suitable detectable groups, such as
an enzyme (e.g., horseradish peroxidase), a member of a binding
pair such as biotin or avidin, a radioactive group or a fluorescent
group such as green fluorescent protein, also in accordance with
known techniques, typically depending upon the immunoassay format
in which the antibody is used.
5. Immunoassay Methods.
The present invention provides a method for detecting incomplete
deprotection of a synthetic oligonucleotide (including aborted
sequences that still contain a protecting group) by immunoassay. In
general, such an immunoassay comprises the steps of: (a) contacting
a synthetic oligonucleotide to an antibody as described above, and
then (b) detecting the presence or absence of binding of said
antibody to said oligonucleotide, the presence of binding
indicating incomplete deprotection of said synthetic
oligonucleotide. Any suitable assay format can be employed,
including heterogeneous and homogeneous immunoassays. For example,
the immunoassay may be an immunoblot-dot assay, or may be a
sandwich assay. The oligonucleotides being tested for deprotection
may be in any suitable form, such as in solution or immobilized on
a solid support.
In a preferred embodiment, the detection method employs a "dip
stick" or the like, in which binding of the antibody to the test
oligonucleotide is compared to binding of the antibody to a set of
known oligonucleotides, all immobilized on a common solid support.
Such an article, as illustrated in FIG. 10, useful for determining
incomplete deprotection of a synthetic oligonucleotide in an
immunoassay, comprises: (a) a solid support (e.g., a nitrocellulose
strip) 25 having a surface portion, said surface portion having at
least two separate discrete regions 26, 27 formed thereon; (b) a
first oligonucleotide bound to one of said separate discrete
regions, said first oligonucleotide having a protecting group bound
thereto (e.g., at least one protecting group); and (c) a second
oligonucleotide bound to another of said separate discrete regions,
said second oligonucleotide not having said protecting group bound
thereto; wherein the nucleotide sequence of said first and second
oligonucleotides are the same. In a preferred embodiment, the
article further comprises (d) a third oligonucleotide bound to
another of said separate discrete regions 28; said third
oligonucleotide also having said protecting group bound to said
first oligonucleotide bound thereto; wherein said third
oligonucleotide is partially deprotected (i.e., has a number of
protecting groups covalently bound thereto which is intermediate
between that bound to the first and second oligonucleotide, e.g.,
at least one, two three or four more protecting groups than the
first oligonucleotide, up to at least 10, 20 or more protecting
groups than the first oligonucleotide); and wherein the nucleotide
sequence of said first, second, and third oligonucleotides are the
same. Of course, still more oligonucleotides carrying varying
numbers of protecting groups may be included on the substrate in
additional separate and discrete locations, if desired. The
discrete regions to which the separate oligonucleotides are bound
may be in any form, such as dots.
6. Affinity Purification Methods.
In addition to immunoassays, the present invention also provides
affinity purification techniques for the separation of fully
deprotected oligonucleotides from partially deprotected (including
fully protected) oligonucleotides (e.g., both oligonucleotides that
have been subjected to a deprotection process to remove the
protecting group, and oligonucleotides that have not). Such a
procedure typically comprises (a) contacting a mixture of protected
and fully deprotected synthetic oligonucleotides to antibodies as
described above, wherein the protected synthetic oligonucleotides
have the organic protecting group for which the antibody is
selective covalently bound thereto, so that the protected synthetic
oligonucleotides bind to the antibody; and then separating said
antibodies from said fully deprotected oligonucleotides. The
antibody may be immobilized on a solid support to facilitate
separation. The protected synthetic oligonucleotide may be a
partially protected synthetic oligonucleotide, or a fully protected
synthetic oligonucleotide that has not undergone deprotection. Any
separation format may be used, including but not limited to
affinity chromatography.
7. Production of Antibodies.
A method of making an antibody that specifically binds to a
synthetic oligonucleotide having a organic protecting group
covalently bound thereto, which antibody does not bind to the said
synthetic oligonucleotide when said organic protecting group is not
covalently bound thereto, comprises the steps of: (a) synthesizing
the synthetic oligonucleotide on a solid particulate support (and
preferably covalently bound thereto, e.g., with a succinyl linker)
with the organic protecting group covalently bound to said
synthetic oligonucleotide; and then, without removing the
oligonucleotide from said solid support; and (b) immunizing an
animal with the synthetic oligonucleotide bound to the solid
support in an amount sufficient to produce the antibody. In
addition, a single nucleotide can be bound to the solid particular
support with the organic protecting group bound thereto, and used
as described hereinabove.
The synthesis step may be carried out on the solid support in
accordance with known techniques. The solid support may be in
particulate form prior to synthesis, or may be fragmented into
particles after synthesis. In general, the solid supports are
beads, which may be completely solid throughout, porous, deformable
or hard. The beads will generally be at least 10, 20 or 50 to 250,
500, or 2000 .mu.m in diameter, and are most typically 50 to 250
.mu.m in diameter. Any convenient composition can be used for the
solid support, including cellulose, pore-glass, silica gel,
polystyrene beads such as polystyrene beads cross-linked with
divinylbenzene, grafted copolymer beads such as
polyethyleneglycol/polystyrene, polyacrylamide beads, latex beads,
dimethylacrylamide beads, composites such as glass particles coated
with a hydrophobic polymer such as cross-linked polystyrene or a
fluorinated ethylene polymer to which is grafted linear
polystyrene, and the like. Where separate discrete solid supports
such as particles or beads are employed, they generally comprise
from about 1 to 99 percent by weight of the total reaction
mixture.
In a preferred embodiment, the synthesizing step is followed by the
step of fragmenting the solid support (e.g., by crushing) prior to
the immunizing step. Polyclonal antibodies may be collected from
the serum of the animal in accordance with known techniques, or
spleen cells may be collected from the animal, a plurality of
hybridoma cell lines produced from the spleen cells; and then a
particular hybridoma cell line that produces the antibody isolated
from the plurality of hybridoma cell lines.
A particular protocol for the production of antiserum/polyclonal
antibodies and monoclonal antibodies against protecting groups used
in nucleic acid and other synthesis typically involves the
following steps: (a) preparation of oligonucleotides and others
that contain or do not contain protecting groups; (b) immunization
of animals with those preparations; (c) screening of animals to
identify those that exhibit antibodies against protecting groups;
(d) production of monoclonal antibody by classical fusion method;
(e) optionally, production of scFab, Fab fragments and whole
antibody molecules by antibody engineering; and (f) evaluation and
characterization of monoclonal antibodies against the protecting
groups. Each of these steps is discussed in greater detail
below.
Synthetic oligonucleotides that contain protecting groups can be
synthesized in a variety of ways known to those skilled in the art.
For example, protecting groups can be attached to individual
nucleotides that are linked to controlled pore glass (CPG) beads.
An example is:
CPG bead - - - dT (only with DMT group).
In the alternative, protecting groups may be attached to
oligonucleotide chains that are linked to CPG beads. Examples
include:
Pac-dA - - - Pac-dA - - - CPG beads with Bz-dC and Ibu-dG;
Ipr-Pac-dG - - - Ipr-Pac-dG - - - CPG beads with Bz-dC and
Ibu-dG;
Ac-dC - - - Ac-dC - - - CPG beads with Bz-dC and Ibu-dG;
dmf-G - - - dmf-G - - - CPG beads with Bz-dC and Ibu-dG; and
mixtures of the four oligonucleotides described above.
In another alternative, protecting groups may be attached to
oligonucleotide chains that are partially deprotected (the
procedure for deprotection will be described bellow). Examples
include:
Poly dT20mers (only with DMT group);
Poly dT20mers (only with cyanoethyl groups);
Poly Ibu-dG 20mers (partially deprotected);
Poly Ipr-Pac-dG 20mers (partially deprotected);
Poly Bz-dC 20mers (partially deprotected);
Poly Pac-dA 20mers (partially deprotected); and
Poly Ac-dC 20mers (partially deprotected).
Synthetic oligonucleotides prepared as described herein may be
partially deprotected as follows: (a) add 30% ammonium hydroxide
solution to synthetic polynucleotides, then incubate at room
temperature for different time periods (5, 10 and 30 min); (b) take
the ammonium solution of treated oligomers and add into 1:1 diluted
acetic acid pre-cooled at 4.degree. C. and according to 1:4 ratio
of ammonium to acetic acid; (c) keep samples in ice bath for 30
min; (d) dry samples with speed-Vac; (e) dissolve the dried pellets
in water; (f) desalt samples with Sephadex G-25 column; (g) dry
samples with speed-Vac; and (h) dissolve the desalted samples in
water.
Synthetic oligonucleotides prepared as described herein may be
completely deprotected by any suitable technique. One particular
technique is as follows: (a) add 30% ammonium hydroxide solution to
synthetic oligonucleotides, then incubate at 65.degree. C. for 6
hrs; (b) dry samples with speed-Vac; (c) dissolve the dried pellets
in water; (d) desalt samples with Sephadex G-25 column; and (e) dry
samples with speed-Vac; (f) redissolve desalted samples in
water.
Partially and completely deprotected oligonucleotides may be
characterized for further use or to verify procedures by any
suitable means, including but not limited to gel electrophoresis,
urea-acrylamide gel electrophoresis, 5' end labeling with T4
polynucleoide kinase, HPLC analysis, mass spectrometry, etc.
Suitable animals can be immunized with the oligonucleotides
described above by parenteral injection of the oligonucleotide in a
suitable carrier, such as sterile saline solution. Injection may be
by any suitable route, including but not limited to subcutaneous,
intraperitoneal, intravenous, intraarterial, intramuscular, etc.
Suitable animals are typically mammals, including mice, rabbits,
rats, etc.
In a particular embodiment, for the production of monoclonal
antibodies, young female BALB/c mice are used, and the time course
of injection of the antigen material is:
TABLE-US-00002 first day initial injection 14th day first boosting
28th day second boosting 4 day before fusion final boosting
Additional injections may be employed if desired. The antigen
amount may be 50 .mu.g or 100 .mu.g of oligonucleotides unprotected
(for control antibody) or protected, for each mouse per time. When,
as preferred, beads or other solid support used as the support for
oligonucleotide synthesis are injected into the animal, the beads
or particles are suspended in water, then injected into mice. If a
nucleotide solution is used, then the solution is mixed with
complete or incomplete Freund's adjuvant and injected into
mice.
Polyclonal antibodies can be harvested from animals immunized or
innoculated as described above in accordance with known techniques,
or spleen cells harvested from the animals, hybridoma cell lines
produced from the spleen cells, and the hybridoma cell lines
screened for the production of desired antibodies, also in
accordance with known techniques.
Oligonucleotides that contain or do not contain biotin molecules at
3' or 5' ends (for ELISA assay as described below) may be
synthesized in accordance with standard techniques. Examples
are:
Poly Ibu-dG 20 mers (with or without biotin);
Poly Ibu-dA 20 mers (with or without biotin);
Poly Ibu-dC 20 mers (with or without biotin);
Poly Ipr-Pac-dG 20 mers (with or without biotin);
Poly Bz-dC 20 mers (with or without biotin);
Poly Bz-dA 20 mers (with or without biotin);
Poly dT 20 mers (with or without biotin);
Poly Pac-dA 20 mers (with or without biotin);
Poly Ac-dC 20 mers (with or without biotin); and
Poly dmf-G 20 mers (with or without biotin).
Antibodies produced as described above may be characterized by any
suitable technique to determine the binding properties thereof,
including but not limited to Western blot and immunodot-blot.
In addition to the use of polyclonal and monoclonal antibodies, the
present invention contemplates the production of antibodies by
recombinant DNA, or "antibody engineering" techniques. For example,
mRNA isolated from hybridoma cells may be used to construct a cDNA
library and the sequence encoding whole antibody or antibody
fragments (e.g., scFab or Fab fragments) isolated and inserted into
suitable expression vector, and the expression vector inserted into
a host cell in which the isolated cDNA encoding the antibody is
expressed.
Monoclonal Fab fragments may be produced in Escherichia coli by
recombinant techniques known to those skilled in the art. See,
e.g., W. Huse, Science 246, 1275-81 (1989).
8. Screening of Antibodies.
Screening sera and hybridoma cell culture media for protecting
group specific antibodies may be carried out as follows:
A. Sera
1. Pre-immune (prior to immunization) sera are collected by
standard means from the mice to be inoculated with protecting group
conjugated to a solid support (directly or through an
oligomer).
2. Post-innoculation sera are also collected.
3. An ELISA assay is performed in which the specific protecting
group remains on a biotinylated oligonucleotide conjugated to the
microtiter plate. Other microtiter plate wells contain control
oligomers that have no protecting groups, or oligonucleotides with
other protecting groups. The secondary antibody is a goat
anti-mouse IgG with a conjugated phosphotase for visualization of
antibody
4. Those mice that have positive activity against the specific
protecting group are boosted and sacrificed for the production of
hybridomas.
B. Hybridoma Cell Culture Media
1. Approximately 1000 cultures are generated from each spleen
hybrid cell production.
2. Cultures are grown in microtiter plate wells, 96 well
plates.
3. Culture medium is removed from each well and used in ELISA
assays as described above in which each of the .about.1000
microtiter plate wells contain the protected oligonucleotide
conjugated to the plate.
4. Those cultures producing antibody that has positive activity are
transferred to larger culture wells, 24 well microtiter plates.
5. Culture media from the larger cultures are re-tested for
activity against the protecting group and are also assayed for
specificity; ie. controls of no protecting group and of other
protecting groups.
6. Those cultures that are positive are cloned out (diluted),
re-tested and cloned out again to the point that each final culture
must be the result of one cell; ie. mono-culture. Media from these
final cultures are thoroughly assessed for specificity and
affinity. Specificity and affinity are assessed using a dot-blot
assay.
C. Dot-Blot assays in lieu of ELISA assays
1. Antibodies against some protecting groups are not tractable to
being tested in the microtiter plate well environment and must be
tested using a dot-blot assay. One example is the 5'-terminal
protecting group, dimethyl-trityl (DMT).
2. The Dot-blot assay on a nitrocellulose membrane is accomplished
as described elsewhere in the application for most purposes.
However, this is not possible in assessing antibody production by
.about.4000 microtiter well cultures with little media available.
Thus, a novel adaptation has been developed.
a) The protected oligonucleotide is attached in dots to the
nitrocellulose using UV-crosslinking. With DMT, the presence of the
5'-DMT on the membrane is confirmed by treatment of a dot with mild
acid--the dot turns yellow-orange. The presence of the 3'-biotin
can be confirmed with a commercial avidin stain.
b) The membrane is blocked (see dot-blot assay).
b) The dry membrane dots are carefully marked (pencil) and
"punched" out of the membrane.
c) Individual dots are added to the cell culture media in
individual micortiter plate wells and incubated.
d) The individual dots are removed and passed on through the
washing, secondary antibody, phosphotase reaction and color
development using microtiter plate wells with the appropriate
reagents.
e) Those dots that are positive are related back to the original
microtiter plate well cultures from which the small amount of
culture media was obtained.
f) Further culturing and cloning is accomplished as described in
B.
9. Testing of Microarrays.
The present invention may be used to test or screen
oligonucleotides that are immobilized on a solid support such as a
microarray for insufficient deprotection or elongation of the
oligonucleotides synthesized thereon.
Solid supports used to carry out the present invention are
typically discrete solid supports. Discrete solid supports may be
physically separate from one another, or may be discrete regions on
a surface portion of a unitary substrate. Such "chip-type" or
"pin-type" solid supports are known. See, e.g., U.S. Pat. No.
5,143,854 to Pirrung; U.S. Pat. No. 5,288,514 to Ellman (pin-based
support); U.S. Pat. No. 5,510,270 to Fodor et al. (chip-based
support). Additional non-limiting examples of oligonucleotide
arrays which may be used to carry out the present invention, and
methods of making the same, include but are not limited to those
described in U.S. Pat. Nos. 5,631,734; 5,599,695; 5,593,839;
5,578,832; 5,510,270; 5,571,639; 6,056,926; 5,445,934; and
5,703,223. Such devices may be used as described therein to carry
out the instant invention.
The solid support or substrate from which the array is formed may
be comprised of any suitable material, including silicon. The
oligonucleotides may be polymerized or grown in situ from monomers
(or individual nucleotides) in situ on the microarray (in which
case none of the currently available techniques for detecting
protecting groups would be useful for detecting incomplete
deprotection or elongation of the oligonucleotides on the array, as
one cannot pass the solid support through an analytical device) or
the oligonucleotides may be polymerized separately and then linked
to the appropriate regions of the solid support. The array may
include any number of different oligonucleotides in different
separate and discrete regions thereon, examples including arrays of
at least 1,000, at least 2,000, at least 10,000, or at least 20,000
different oligonucleotides in different separate and discrete
regions.
In general, a method of screening an oligonucleotide array for
insufficient deprotection or insufficient elongation of
oligonucleotides therein comprises the steps of:
(a) providing an oligonucleotide array as described above;
(b) providing an antibody as described above (that is, an antibody
that specifically binds to a synthetic oligonucleotide having an
organic protecting group covalently bound thereto, which antibody
does not bind to said synthetic oligonucleotide when said organic
protecting group is not covalently bound thereto). Preferably the
antibody is one that specifically binds to an oligonucleotide
having a protecting group, where the protecting group was employed
in the course of the organic synthesis of oligonucleotides carried
by that array. Then;
(c) contacting said oligonucleotide array to said antibody to
thereby detect the presence of insufficient deprotection or
insufficient elongation of oligonucleotides therein. Such
detection, which may be qualitative or quantitative, may be carried
out by any suitable immunoassay technique as described above.
In the method, steps (b) to (c) may be repeated at least once, with
a different antibody on each repetition, so that a plurality of
different protecting groups which may be present on
oligonucleotides in the array may be detected.
Preferably, once insufficient deprotection (the presence of
protecting groups) in oligonucleotides in one or more (e.g.,
plurality) of the separate and discrete regions is detected, the
method further comprises generating a record or indicia recording
the presence of insufficient deprotection or insufficient
elongation of oligonucleotides in the least one separate and
discrete location (or plurality of separate and discrete locations)
on the array. The indicia may be a qualitative or quantitative
indicia of insufficient deprotection (including insufficient
elongation).
The foregoing methods provide a correctable oligonucleotide array
as illustrated in FIG. 11. The array comprises, in combination:
(a) a substrate 30 having a plurality of different oligonucleotides
immobilized thereon, with the different oligonucleotides
immobilized in different separate and discrete locations 31 on said
substrate; and
(b) a plurality of indicia associated with said array, these
indicia recording the presence of insufficient deprotection or
insufficient elongation of a plurality of different
oligonucleotides, said different oligonucleotides located in
separate and discrete locations on said array. These indicia may be
printed in a region of the array 32 by a technique such a
microlithography, printed on conventional medium such as paper and
shipped with the array, stored in a memory or memory device
connected to or formed on the array chip (which may be incorporated
at location 32), provided in a separate data or computer file which
may be provided on a computer-readable medium such as a floppy
diskette or CD-ROM, stored on a web site on the world wide web for
downloading by the end user of the array, etc. When the indicia are
provided in a separate data file, the array preferably further
includes an identifier such as a code number formed on, connected
to or associated with the array (e.g., printed on a package
containing the array, or on an information sheet packaged with the
array, and/or printed directly on the array). The identifier may
then be associated with the separate indicia (e.g., printed on a
data sheet, used as a pass-word, file identifier and/or access code
for the computer file, etc.) to insure the correct indicia
containing the record of insufficient deprotection and/or
elongation are ultimately associated with the array by the ultimate
end user of the array.
A data device or memory device connected to the array may be
carried out in accordance with known techniques, as described in
U.S. Pat. Nos. 5,925,562; 6,017,496; 5,751,629; and 5,741,462, and
such devices used as described therein to carry out the instant
invention.
The end user of the array may utilize the indicia described above
to compensate for insufficient deprotection or insufficient
elongation of oligonucleotides on said array in a method
comprising:
(a) providing a substrate as described above.
(b) providing at least one, or a plurality of, indicia associated
with said array as described above.
(c) providing a test compound. The test compound may be a member of
a library of test compounds, and may be any suitable compound such
as a protein, peptide or oligonucleotide (e.g., a DNA or RNA, such
as mRNA); and then
(d) detecting the binding of said test compound to at least one of
said plurality of different oligonucleotides (e.g., by contacting
the test compound to the array); and then
(d) detecting determining the degree of binding (including simply
the presence or absence of binding) of the test compound to one or
more oligonucleotides on the array from (i) said detected binding
and (ii) said indicia recording the presence of insufficient
deprotection or insufficient elongation. Thus, insufficient
deprotection or insufficient elongation of oligonucleotides in one
or more locations in the array may be compensated for during the
determining step. Such compensation may be achieved by any means,
including ignoring particular separate and discrete regions on the
array (e.g., in favor of other separate and discrete regions of the
array that contain the same oligonucleotide). In another example,
if one or more locations contain insufficient deprotection or
elongation such that binding to those locations is reduced, the
binding data derived from an experiment with that array can be
adjusted upwards for those locations to indicate greater binding
than that which would otherwise be indicated without the control
made possible by the recorded indicia. The detecting or determining
step may be carried out by any suitable means, such as generating a
color indication of degree of binding, generating a numeric
indication of degree of binding, generating a graphic or other
symbolic indication of degree of binding, etc. The degree of
binding may be an indication of binding is binding affinity,
binding amount, or both binding affinity and binding amount, but is
typically an indication of the amount of test compound that binds
to a particular separate and discrete region of the array.
The present invention is explained in greater detail in the
following non-limiting Examples.
Example 1
Synthesis of Oligonucleotides
Synthesis was performed on an ABI DNA/RNA Synthesizer, Model 394
(PE Biosystems, 850 Lincoln Centre Drive, Foster City, Calif.
94404) according to manufactories protocol. Slightly modified 1
micromolar scale cycle was used during synthesis (see
manufacturer's instructions). The primary starting materials (and
suppliers/manufacturers in parentheses) were as follows: Activator
(0.45 M tetrazole in acetonitrile), CAP A (acetic
anhydride/tetrahydrofuran/2,6 lutidine), CAP B (N-methyl
imidazole/tetrahydrofuran) and oxidizer (0.02 M
iodine/pyridine/THF/H2O) (Prime Synthesis) Pac-dA
(5'-dimethoxytrityl-N-phenoxyacetyl-2'-deoxyAdenosine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen
Research) Ipr-Pac-dG
(5'-dimethoxytrityl-N-p-isopropyl-phenoxyacetyl-2'-Guanosine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen
Research) Ac-dC (5'-dimethoxytrityl-acetyl-2'-deoxycytidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen
Research) dmf-G (5'-dimethoxytrityl-dimethylformamidine-Guanosine,
2'-O-TBDMS-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
(Glen Research) Bz-dC - - - CPG beads
(5'-dimethoxytrityl-N-benzoyl-2'-deoxycytidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-succinyl
linker-beads (3000 Ang) (CPG Inc.) Ibu-dG - - - CPG beads
(5'-dimethoxytrityl-N-isobutyl-2'-deoxycytidine,
3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite-succinyl
linker-beads (3000 Ang) (CPG Inc.)
The following compounds were synthesized, the compounds being
linked to beads as shown: Pac-dA - - - Pac-dA - - - Bz-dC - - -
succinyl linker - - - Beads Pac-dA - - - Pac-dA - - - Ibu-dG - - -
succinyl linker - - - Beads Ipr-Pac-dG - - - Ipr-Pac-dG - - - Bz-dC
- - - succinyl linker - - - Beads Ipr-Pac-dG - - - Ipr-Pac-dG - - -
Ibu-dG - - - succinyl linker - - - Beads Ac-dC - - - Ac-dC - - -
Bz-dC - - - succinyl linker - - - Beads Ac-dC - - - Ac-dC - - -
Ibu-dG - - - succinyl linker - - - Beads dmf-G - - - dmf-G - - -
Bz-dC - - - succinyl linker - - - Beads dmf-G - - - dmf-G - - -
Ibu-dG - - - succinyl linker - - - Beads
The foregoing compounds were administered directly to animals as an
immunogen, without separating the oligonucleotide from the solid
support, for the production of antibodies, as further described in
Example 2 below.
Example 2
Innoculation of Animals
Female BALB/c mice of eight to twelve weeks old were purchased from
Charles River, Raleigh, N.C., USA. The mice were housed in cases
with filter caps.
After oligonucleotide chain synthesis was completed as described in
Example 1, the beads with nucleotides were gently crushed by
hand-pressuring the glass plates, between which beads were
positioned.
5 .mu.M each eight oligonucleotides mentioned above were mixed in 4
ml PBS (150 mM sodium chloride in 100 mM phosphate buffer, pH
7.2).
The mixture was thoroughly vortexed suspending the crushed beads.
150 .mu.L of the vortexed mixture was taken and added into 300
.mu.L of PBS in a syringe. Just before injection, the solution
containing beads was mixed again by shaking the syringe to suspend
the broken beads. Then 150 .mu.L or 300 .mu.L of well-mixed
solution was injected into mouse peritoneal cavity. This procedure
was used for the first injection and the following boosts.
Injection Time Schedule:
TABLE-US-00003 Injection Date (day) first 0 second 14th third 28th
4th 42nd 5th 56th 6th 70th 7th 84th 8th 98th 9th 112th 10th 138th
11th (final, 4 day before fusion) 142nd
Four days after the final injection, spleen cells are harvested
from the animals and fused with myeloma cells (P3x.63.Ag8.653) in
accordance with known techniques to produce hybridoma cell lines,
which are then screened to determine the binding characteristics as
described below to isolate particular cell lines that produce the
desired antibody of the invention.
Example 3
Immunodot-Blot Assay for Antibody Characterization
The Immunodot-blot assay involves UV cross linking of
oligonucleotides onto membrane paper, and is directly applicable to
a test kit for detection, identification and quantifying the
protecting groups on product oligomers. This procedure may be
carried out as follows: (a) wet membrane paper with TBS (10 mM
Tris, pH 7.2; 150 mM NaCl); (b) blot oligonucleotides to be tested
onto membrane paper under vacuum; (c) UV cross link nucleotide onto
membrane paper; (d) block membrane paper with 1% casein-TBST (TBS
plus Tween 20, 0.1% by volume) at room temperature for 2 hr or
4.degree. C. overnight; (e) wash membrane with TBST 3 times, each
for 15 min; (f) form antigen-antibody complex by incubation of
plate with sample be tested (diluted in 1% casein-TBST) at room
temperature for 1 hr; (g) wash as above; (h) react with second
antibody conjugate (diluted in 1% casein-TBST) at room temperature
for 1 hr; (i) wash as above; (j) develop color reaction by
incubation of membrane with substrate solution.
Example 4
Dot-Blot Assay of Monoclonal Antibody 1H11
Monoclonal antibody 1H11, produced as described in Example 2 above,
was characterized by a dot-blot assay as described in Example 3
above. Results are shown as a bar graph in FIG. 1. In FIG. 1, lanes
(or columns) 1 and 2 represent oligoPac-dA20mers treated with
NH.sub.4OH for 6 hours at 65.degree. C. and 15 minutes at 4.degree.
C., respectively. Columns 3 and 4 represent oligoBz-dC20mers
treated with NH.sub.4OH for 6 hours at 65.degree. C. and 15
minutes, respectively. Columns 5 and 6 represent oligoAc-dC20mers
treated with NH.sub.4OH for 6 hours at 65.degree. C. and 15
minutes, respectively. Columns 7 and 8 represent
oligoIpr-Pac-dG20mers treated with NH.sub.4OH for 6 hours at
65.degree. C. and 15 minutes, respectively. Columns 9 and 10
represent oligoIbu-dG20mers treated with NH.sub.4OH for 6 hours at
65.degree. C. and 15 minutes, respectively. Columns 11, 12 and 13
represent oligodT20mers, completely deprotected, with DMT group
only, and with cyanoethyl group only, respectively. Antibody
activity is given as optical density (479 nm) from ELISA (Example 7
below), and the positive or negative result of the dot blot assay
is given in the open or filled circle appearing over each column in
the bar graph. Note the activity of monoclonal antibody 1H11 in
selectively binding to the oligoIbu-dG20mer in column 10.
Example 5
Dot-Blot Assay of Monoclonal Antibody 7 H3
Monoclonal antibody 7 H3, produced as described in Example 2 above,
was characterized by a dot-blot assay as described in Example 3
above. Results are shown as a bar graph in FIG. 1. In FIG. 1, lanes
(or columns) 1 and 2 represent oligoPac-dA20mers treated with
NH.sub.4OH for 6 hours at 65.degree. C. and 15 minutes at 4.degree.
C., respectively. Columns 3 and 4 represent oligoBz-dC20mers
treated with NH.sub.4OH for 6 hours at 65.degree. C. and 15
minutes, respectively. Columns 5 and 6 represent oligoAc-dC20mers
treated with NH.sub.4OH for 6 hours at 65.degree. C. and 15
minutes, respectively. Columns 7 and 8 represent
oligoIpr-Pac-dG20mers treated with NH.sub.4OH for 6 hours at
65.degree. C. and 15 minutes, respectively. Columns 9 and 10
represent oligoIbu-dC20mers treated with NH.sub.4OH for 6 hours at
65.degree. C. and 15 minutes, respectively. Columns 11, 12 and 13
represent oligodT20mers, completely deprotected, with DMT group
only, and with cyanoethyl group only, respectively. Antibody
activity is given as optical density as described above, and the
positive or negative result of the dot blot assay is given in the
open or filled circle appearing over each column in the bar graph.
Note the activity of monoclonal antibody 1H11 in selectively
binding to the oligoBz-dC20mer in column 4.
Example 6
Western Blot Assay for Antibody Characterization
The Western blot assay involves low voltage transfer of
oligonucleotides from gel to membrane paper and UV cross linking of
oligonucleotides onto the membrane. This assay may be carried out
as follows: (a) cast 15% non-denaturing gel containing 10 mM MgCl;
(b) load oligonucleotides (oligomers) into the wells of the gel;
(c) run gel at 200 voltage in ice bath; (d) transfer
oligonucleotides from gel to membrane paper at 25 voltage for 25
min in ice bath; (e) UV cross link polynucleotides on membrane; (f)
block membrane paper with 1% casein-TBST at room temperature for 2
hr or 4.degree. C. overnight; (g) wash membrane with TBST 3 times,
each for 15 min; (h) incubate samples be tested (diluted in 1%
casein-TBST) at room temperature for 1 hr; (i) wash as above; (j)
incubate membrane with second antibody conjugate (diluted in 1%
casein-TBST) at room temperature for 1 hr; (k) wash as above; and
(l) color-develop by incubation of membrane with substrate
solution.
Example 7
Detection of Antibody Using Biotinylated Polynucleotides as Antigen
and an ELISA Involving Streptavidin-Biotin System
An enzyme-linked immunosorbent assay (ELISA) for the detection of
the antibody is carried out as follows: (a) pre-screen microtiter
plate that is pre-coated with streptavidin; (b) coat the plate with
a preparation of biotinylated oligonucleotide or other materials to
be tested (at 5 .mu.g/ml in PBS)(PBS: 150 mM NaCl, 10 mM Phosphate
buffer, pH 7.4), then incubate at room temperature for 2 hrs; (c)
wash 3 times with 0.1% Tween in PBS (PBST), each for 15 min; (d)
block with 1% casein in PBST at room temperature for hrs or
4.degree. C. overnight; (e) wash as above; (f) form
antigen-antibody complex by incubation of plate with antibody (or
antibodies) at room temperature for 1 hr; (g) wash as above; (h)
react with second antibody-peroxidase conjugate (in 1% casein-PBST)
at room temperature for 1 hr; (i) wash as above; (j) develop color
reaction by adding tetramethylbenzidine (TMB) solution (TMB
solution: 42 mM TMB, 0.004% H.sub.2O.sub.2, 0.1 M acetate buffer,
pH 5.6) and incubating at room temperature for 15 mM, then stop the
reaction with 2 M H.sub.2SO.sub.4; and (k) read absorption value at
469 nm.
Example 8
ELISA and Dot-Blot Assay of Monoclonal Antibody Against Benzoyl,
Isobutryl, and Isopropylphenoxyacethyl
Monoclonal antibodies (mAb) against protecting groups benzoyl (Bz),
isobutryl (ibu), and isopropylphenoxyacethyl (ipr-Pac), produced as
described in Example 2 above, were characterized by a standard
ELISA assay and a dot-blot assay as described in Example 3 above.
An ELISA assay developed with biotinylated nucleic acids of 20
residues each attached to a 96-well microtiter plate demonstrated
the specificity of the antibodies for their respective antigens.
FIG. 3A, FIG. 4A, and FIG. 5A show results for monoclonal
antibodies against Bz, ibu, and ipr-Pac, respectively. The figures
show completely deprotected (<1% Bz remaining) homopolymers of
dC residues, designated oligo dC(Bz), ie. originally protected with
Bz (lane 1, open bar), protected (>97% Bz remaining) oligo Bz-dC
(lane 2, shaded bar), completely (<1% ipr-Pac remaining)
deprotected oligo dG(ipr-Pac) (lane 3), protected (>76% ipr-Pac)
oligo ipr-PacdG (lane 4), completely (<1% ibu remaining)
deprotected oligo dG(ibu) (lane 5), protected (>91% ibu
remaining) oligo ibu-dG (lane 6), and completely deprotected oligo
dT (lane 7). The dT polymer had but one protecting group,
dimethyltrityl (DMT) that was removed from the 5'OH of the
5'-terminal residue with mild acid. Finally, lane 8 of shows oligo
dT with DMT remaining.
Dot-Blot assays of anti-Bz mAb, anti-ibu mAb, and anti-ipr-Pac mAb
activities were performed in which the 20mer DNAs were linked to
nitrocellulose membrane by UV. The amounts of 20mer DNA applied to
the membrane are shown to the right of FIG. 3B, FIG. 4B, and FIG.
5B and demonstrate the level of sensitivity of the assay. The DNAs
used to test anti-Bz mAb were those described for the ELISA plus
deprotected oligo dA(Bz), protected oligo Bz-dA, oligo dC(ibu),
oligo ibu-dC, oligo dA(ibu) and oligo ibu-dA. FIG. 3B shows that
the anti-Bz mAb recognized the protecting group on dA and dC. The
DNAs used to test anti-ibu mAB were those described for the ELISA
plus protected oligo ibu-dA, deprotected oligo dA(ibu), oligo
ibu-dC, oligo dC(ibu) and all are noted at the top of the dot-blot.
FIG. 4B shows that the anti-ibu mAb recognized ibu on dG, the most
common use of the protecting group, but also on dA. The DNAs used
to test anti-ipr-Pac mAb were those described for the ELISA plus
protected oligo ibu-dA, deprotected oligo dA(ibu), oligo ibu-dC,
oligo dC(ibu), oligo Bz-dA, oligo dA(Bz) and all are noted at the
top of the dot-blot. FIG. 5B shows that the anti-ipr-Pac mAb
recognized ipr-Pac on dG, the most common use of the protecting
group, but also on dA and dC. The mAb also recognized the ibu
protecting group (ibu-dG, ibu-dA and ibu-dC). This cross-reactivity
indicates that the antibody was highly selective in its
identification of a chemistry common to both ipr-Pac and ibu,
possibly CH(CH.sub.3).sub.2. Thus the anti-ibu and anti-iprPac mAbs
could be used in combination to identify the protecting group
remaining on an oligo.
Greater amounts of DNA were tested in a dot blot assay of anti-ibu
mAb (FIG. 4C). The results of this experiment demonstrated that the
ibu protecting group was recognized by the mAb no matter which
nucleobase was protected.
FIG. 3C, FIG. 4D, and FIG. 5C demonstrate that partially
deprotected oligomers can be re-treated to remove the remaining
protecting groups, and re-tested with mAb. FIG. 3C shows that
anti-Bz mAb recognized re-deprotected oligomer oligo Bz-dC (center
column). Likewise, FIG. 4D shows that anti-ibu mAb recognized
re-deprotected oligomer oligo ibu-dG (center column) and FIG. 5C
shows that anti-ipr-Pac mAb recognized re-deprotected oligomers
oligo ipr-Pac-dG and oligo ibu-dG (columns second from left and
forth from left, respectively). Thus, this approach is applicable
to quality control without having to discard expensive nucleic acid
samples.
An RNA standard with protecting groups Bz, ibu and ipr-Pac was
synthesized and assayed for identification of the protecting groups
with the mAb against Bz (FIG. 3D), ibu (FIG. 4E), and ipr-Pac (FIG.
5D). Dot-blot assays clearly show that the monoclonal antibodies do
not differentiate RNA from DNA. Although there was a higher
background signal with RNA than with DNA, there was a significant
distinction between RNA with and without protecting groups,
especially at the lower amounts of RNA. The amount of RNA on the
membrane was estimated from the optical absorbance of the
sample.
Example 9
mAb Dot-Blot Assay of Protecting Groups Vs HPLC
Dot-blot detection of Bz groups remaining on a standardized 20mer
oligo dC molecule were performed as described in Example 3.
Completely deprotected and the untreated oligo dC 20mers were
analyzed for the Bz protecting group using a totally independent
and different quantification method. The two oligomers were
hydrolyzed to the constituent nucleosides and then their nucleoside
composition identified and quantified using a recognized high
performance liquid chromatography (HPLC) method with concentrated
samples. Because of the lack of sensitivity, HPLC detection
required 50-100 fold the amounts of Bz-dC used in the mAb assays
(see FIG. 7). FIG. 6A shows the result of anti-Bz mAb tested
against nmole amounts of Bz groups on protected oligo Bz-dC (right
column) and the same nmole amounts of Bz- on Bz-dC (left column).
Each amount of Bz-dC oligo was diluted with completely deprotected
dC oligo of the same length (20mer) to demonstrate the sensitivity
of the mAb detection even in the presence of 2500-fold dC (ie.
0.04%). The mAb assay demonstrated that the mAb could detect the Bz
group on DNA even in the presence of a 2500-fold excess of dC in
DNA.
The dot-blot shown in FIG. 6A was subjected to densitometry to
quantitate the mAb response. After background subtraction, the
remaining density was plotted as a function of Bz groups in oligo
Bz-dC determined by HPLC (FIG. 6B). The data indicated that the
high sensitivity of the anti Bz mAb detection was linear in 0.1-1.0
nmol range.
Next, it was determined whether the mAb response could be enhanced
with an increase in the amount of DNA on the dot-blot membrane. The
amount of Bz was determined by standard HPLC methods. This
experiment showed that detection of the Bz protecting group in a
mixture of the protected sample with the deprotected sample at a
ratio of 1/2500 could be enhanced by increasing the amount of DNA
on the membrane, though the ratio was maintained (FIG. 6C).
Finally, experiments were conducted to show a direct comparison of
the mAb and HPLC detection of Bz. Anti-Bz mAb was utilized in a
dot-blot assay to detect Bz on dC in the oligo Bz-dC (20mer). The
density response of the Bz group detected Bz by the mAb assay and
quantified by densitometry was plotted against the amount of Bz in
the DNA on each dot (FIG. 7A). The amount of Bz in the DNA was
calibrated by digestion of a large amount of DNA and analysis by
HPLC identification and quantification of the Bz-dC mononucleoside.
For HPLC experiments, three samples of Bz-dC oligo were hydrolyzed
and analyzed for composition by HPLC. The response of the UV-diode
array detector was plotted against the amount of Bz in the samples
(FIG. 7B). The sample amounts were determined by comparison to
samples "spiked" with known amounts of Bz-dC. The amounts of Bz-dC
added to samples as spikes were from a weighed stock of Bz-dC.
Thus, the HPLC response was calibrated with known amounts of Bz-dC.
The results of these experiments show that the detection of Bz by
anti-Bz mAb was within the pmole range whereas HPLC detection of Bz
was limited to the nmole range.
Example 10
Detection of Remaining Protecting Groups in Commercial Samples
A blind study was conducted to demonstrate the detection of
remaining protecting groups in commercial samples by mAb. The
purpose of the this experiment was to determine if protecting
groups could be detected and identified with mAb technology in
presumably completely deprotected samples that had been treated as
commonly accomplished in the oligo synthesis industry. The nature
of the protecting groups used by eight selected companies was not
known, thus the experiment was a blind study. Two 20mer oligos
(oligo dA-dC and oligo dG-dT) from each of the eight companies were
ordered to be synthesized and deprotected, and salt removed under
as identical conditions as possible. The oligos were shipped by
express mail, as is often the case, and then subjected to mAb
analysis by dot blot. The dA-dC oligo from one company (#6), and
possibly a second (#2), had remaining Bz protecting groups as
determined by anti-Bz mAb testing (FIG. 8A). The dG-dT oligos from
two companies (#2 and #6) had ipr-Pac protecting groups remaining
as determined by anti-ipr-Pac mAb (FIG. 8B). The remaining
protecting groups in the commercial samples were confirmed by
increasing amounts of sample and further deprotection and
re-analyses. The oligo dA-dC samples from companies #2 and #6 were
tested in higher amounts to confirm the presence of the Bz
protecting group. In addition, the samples were treated to remove
the remaining protecting groups using a standard protocol. The
re-analysis after further deprotection indicated that the groups
were now removed (FIG. 8C). This also demonstrates that expensive
nucleic acid samples can be re-treated to remove protecting groups
and that they need not be discarded. The oligo dG-dT samples were
re-treated to remove remaining protecting groups and re-analyzed
with anti-ipr-Pac mAb with the result that the ipr-Pac group could
be removed without sacrificing the DNA (FIG. 8D).
Example 11
Polyclonal Antibody Against Dimethyltrityl
Production and analyses of polycolonal antibody against the 5'
terminal protecting group, dimethyltrityl (DMT) were as described
in Example 2. Four mice were inoculated with DMT and sera were
drawn from the mice after some weeks of boosting with antigen.
DMT[DMT-OH], three DMT at the 5'-end of the deoxynucleotide trimer
d(T).sub.3 [(DMT).sub.3-d(T).sub.3], three DMT at the 5'-end of the
deoxynucleotide 20mer d(T).sub.3 with 3'-biotin
[(DMT).sub.3-d(T).sub.20-biotin], one DMT at the 5'-end of the
deoxynucleotide 20mer d(T).sub.20 with 3'-biotin
[DMT-d(T).sub.20-biotin], the dT 20mer with 3'-biotin
[d(T).sub.20-biotin], one DMT with biotin [DMT-biotin] and
tris-borate saline control were applied to a nitrocellulose
membrane that was then assayed with mouse sera (inoculated mice
#1-4 and a control serum, normal) to assess anti-DMT antibody, mild
acid to reveal presence of the DMT (TBS), and avidin to reveal the
presence of biotin (FIG. 9). Sera from mice #2 and #4 recognized
DMT [as (DMT).sub.3-d(T).sub.3], whereas mice #1, #3, and the
normal mouse did not. Mild acid revealed the presence of DMT as a
yellow color (not visible in figure) and avidin revealed the
presence of biotin.
The foregoing is illustrative of the present invention, and is not
to be construed as limiting thereof. The invention is defined by
the following claims, with equivalents of the claims to be included
therein.
* * * * *